For traditional vaccination, attenuated pathogens are in use to elicit an immune response from the recipient organism that is also active against wild type pathogens. An example for the use of attenuated invasive bacteria as a vaccine is the use of attenuated enteropathogenic salmonella, providing protection against salmonellosis.
Depending on the disease against which immunoprotection is intended, it is desired to elicit a variety of the immune responses such as the stimulation of antibody production and/or T-cell mediated immunity, systemically and/or mucosally. As the quality of these immunoreactions is at least in part dependent on the route of administration, a vector according to the invention can be designed to preferentially elicit a cellular and/or humoral immune response.
Although effective in recognising antigens and marking antigen for subsequent removal, antibodies cannot bind to intracellular antigens, for example to bacteria which are contained within the phagosome of infected cells, or to cytosolic antigens of malignant cells, i.e. tumor cells. This part of the immune response is provided by the cellular immune response, i.e. T-cells recognizing foreign peptide displayed on the outer surface of infected cells in conjunction with MHC-I and/or MHC-II complexes. However, in relation to tumor cells, the cellular immune response against these is often hampered by self-tolerance of the immune system against homologous constituents.
In general, antigen specific cytotoxic T-lymphocytes (CTL), which are one sub-group of T-cells, are activated by interaction with antigen presented on the cell surface in a complex with MHC I, which is the presentation pathway for cytosolic protein of mammalian cells. Accordingly, antigen synthesized by invasive pathogens in the cytoplasm, or antigen synthesized by bacterial vectors based on attenuated invasive bacteria which liberate antigen into the cytoplasm, is efficient in eliciting the cellular immune response by CTL. Following this activation, CTL are immediately directed against infected cells that display the relevant antigenic peptides in complex with MHC I.
Some invasive bacteria, e.g. salmonella, are able to specifically target professional antigen presenting cells (APC) in the infected organism. In the case of salmonella, the bacteria invade the mammalian organism mainly by passage through the M cells of Peyer patches within the small intestiny. Subsequent to passaging M cells, invasive bacteria infect dendritic cells and macrophages present in Peyer patches which are part of the mucosa associated lymphoid tissue. Peyer patches contain an increased number of APC of the gut associated lymphoid tissue, wherein bacteria can persist for days up to several weeks, depending on the degree of their attenuation. APC infected with salmonella can migrate into mesenteric lymph nodes, spleen and liver, effectively transporting invaded bacteria into these tissues. Macrophages which are lysed after infection by invasive bacteria are phagocytosed by dendritic cells.
Some invasive bacteria, e.g. salmonella, are able to replicate within the phagosome of mammalian cells, e.g. macrophages, by secreting a range of proteins and further effector molecules. These secreted molecules for example prohibit the import of NADPH oxidase into the phagosome, effectively preventing an immediate antibacterial response by the macrophages. As a further result of the infection with salmonella, caspase 1 is activated, leading to the secretion of inflammatory cytokines (IL-1 beta, IL-18) and initiating apoptosis of infected cells. Material of apoptotic macrophages which are phagocytosed by dendritic cells enter the presentation pathway of MHC I and MHC II, in turn initiating specific T-cell responses.
Some invasive bacteria, e.g. Listeria monocytogenes and Shigella spp., are able to replicate within the cytoplasm of mammalian cells. Upon lysis, the bacterial content can be released directly into the cytosol of infected cells, providing access to the MHC-I presentation pathway.
It is known that bacterial vaccine strains can induce mucosal and systemic immune responses, e.g. Salmonella administered orally.
Attracted by the efficiency of invasive bacteria to elicit an immune response, attenuated invasive bacteria have been investigated to serve as bacterial vectors for vaccines eliciting an immune response against a peptide foreign to the bacterial vector.
In addition, attenuated invasive bacteria have been used for genetic vaccination and gene therapeutic purposes as carrier vehicles to introduce eukaryotic expression plasmids into mammalian cells. In order to allow transcription of DNA sequences introduced into a mammalian cell, the DNA needs to be transported into the cellular nucleus. One prerequisite for efficient DNA transfer is an efficient invasion of host cells by the bacterial carrier vehicle. After invasion of the host cell, lysis of the bacterial vector to release its contents into the cytoplasm and a subsequent effective translocation of DNA into the nucleus are essential steps for transcription of the eukaryotic expression plasmid.
When using attenuated invasive bacteria as bacterial vectors for the transfer of protein and/or nucleic acid into mammalian cells, it is desirable to control the level of expression of said peptide and/or the copy number of nucleic acids. Up to now most attempts to control the expression of a foreign peptide by bacterial vectors rely on constitutive promoter sequences. Recently, also in vivo inducible promoters isolated from invasive bacteria, e.g. Salmonella, and have been used for this purpose. These promoters are regulated depending on the stage of the infectious cycle of said bacteria. Accordingly, such promoter sequences automatically respond to specific phases of the infectious cycle of the bacterium, so that they cannot be regulated by exerting an external influence. For example, Dunstan et al. (Infection and Immunity, October 1999, pages 5133 to 5141) disclose construction and analysis of expression plasmids for use in the known attenuated vaccine strain Salmonella enterica serovar Typhimurium ΔaroAD as the bacterial vector for a vaccine. The promoter elements were arranged before the structural gene for the C-terminal fragment of the tetanus toxin and luciferase, respectively. The in vivo induction to the promoter elements originating from salmonella and E. coli depends on environmental factors that naturally occur during the infectious cycle. For example, the promoter pagC is induced within the phagosomal compartment of eucaryotic cells as a result of their low concentrations of Mg2+.
Dietrich et al. (Current Opinion in Molecular Therapeutics 5, 1, pages 10-19 (2003)) discuss the release of DNA from Listeria monocytogenes by an active lytic process, achieved by lysis of intracellular bacteria by treatment with antibiotics or, alternatively, by dedicated autolysis of the vector organism. For dedicated autolysis, the Listeria-specific phage lysin gene is suggested for expression under control of a listerial promoter, which is activated upon escape of the bacteria from the host cell phagosome into the cytosol. However, induction of this promoter regulating the lysis gene is only dependent upon the environment of the vector, whereas a control from outside the bacterial vector, i.e. arbitrarily, is impossible.
Jain et al. (Infection and Immunity, 986-989 (2000)) report the expression of lysis genes S and R from page lambda in gram-negative bacteria under the control of an L-arabinose inducible promoter in vitro.
However, in the state of art presently known to the inventors, no hint or assumption can be found that a saccharide inducible promoter functionally linked to a transgene, contained within a bacterial vector by genetic modification, can be controlled by applying a stimulus to an animal that has been treated by administration of the bacterial vector. In detail, the state of art does not suggest that induction of a transgene may be controlled under circumstances where the inductor cannot be delivered to the bacterial vector directly, i.e. the vector is located within a tissue, an organ or an animal at a distance from the localisation and/or in a distance from the point in time of administration of the inductor.